Pyrosequencing reveals the impact of foliar fungicide application to chickpea on root fungal communities of durum wheat in subsequent year

Frequent application of foliar fungicide is essential for chickpea production due to the susceptibility of this plant to ascochyta blight. Chlorothalonil, pyraclostrobin, and boscalid are commonly used to control the disease in Saskatchewan. While fungicides are meant to target specific fungal pathogens, they may impact non-target organisms and alter soil microbial community structure. The effects of the typical 5-time foliar fungicide application program to chickpea CDC Vanguard on the fungal communities associated with seminal and adventitious roots of the following durum wheat crop were studied in a 2 yr field experiment. Root fungal communities were characterized through analysis of the ITS1 region of root metagenomic DNA at the genus level. One hundred and seven fungal genera were detected in durum wheat roots. Fusarium was predominant in both years. A three-way interaction of fungicide application, root type and year on fungal community structure was detected. Unlike Fusarium, the relative abundances of the genera Olpidium, Alternaria, and Cryptococcus were greater in 2010, a very wet year. Fungicide application to chickpea increased the relative abundance of Fusarium in the seminal roots of a subsequent durum crop in 2009, but did not affect the relative abundance of Fusarium in 2010. We could not detect a significant impact of fungicide application to chickpea on durum wheat yield in the subsequent year. The effect of changes in root fungal communities on durum wheat grain yield is discussed.     

Differential mutagenicity of streptozotocin analogs of the carbohydrate moiety

The mutagenicity of streptozotocin (SZN), 8 of its analogs and N-msthyl-N-nitrosourea (MNU) were compared in Salmonella typhimurium. SZN and its analogs carry MNU attached to the carbohydrate moiety at the C-2 position. The C-1 analogs tested were α- and β-methyl-SZN, α-ethyl-SZN, β-propyl-SZN, α- and β-butyl-SZN; in 2 analogs glucose was replaced by α- or β-inositol. When the ability of these compounds to revert the hisG46 auxotroph was compared, they fell into 4 groups which differed by about 10-fold in mutagenicity from one another. The most mutagenic was (i) SZN, followed by (ii) β-methyl-SZN; (iii) α-methyl-SZN, α-ethyl-SZN, β-propyl-SZN, α- and β-butyl-SZN; (iv) α and β-inositol-MNU. These results suggest that the presence of the glucose moiety is conducive to a high level of mutagenicity of SZN. Alterations of the glucose moiety by addition of larger alkyl groups, especially in the α position lead to decreased mutagenicity. The least mutagenic analogs are those in which the glucose moiety is replaced by inositols.The mutagenicity of SZN, β-methyl-SZN and of β-butyl-SZN was also compared in a mouse tissue-mediated assay. SZN was about 500-fold more mutagenic than its β-methyl analog, while the β-butyl analog was not mutagenic.Depletion of SZN and 4 of its analogs from the medium in presence of bacteria was determined spectrophotometrically. The more mutagenic compounds were depleted more rapidly but the quantitative differences in mutagenicity between these compounds could not be accounted for by depletion alone.     

In vitro synthesis of a mutagenic azide metabolite by cell-free bacterial extracts

Cell-free extracts of Salmonella typhimurium synthesize a mutagenic azide metabolite from sodium azide and O-acetylserine. S. typhimurium mutant DW379 (O-acetylserine sulfhydrylase-deficient) extracts were neither able to carry out this reaction nor produce the mutagenic azide metabolite in vivo. The in vitro reaction was inhibited by sulfide but not by l-cysteine. The catalytic activity responsible for the mutagenic metabolite synthesis was stable to brief heating up to 55°C and had a pH optimum between 7–7.4. These results suggest that the enzyme O-acetylserine sulfhydrylase catalyzes the reaction of azide with O-acetylserine to form a mutagenic azide metabolite.     

Mutagenic effects of allylisothiocyanate inEscherichia coli WP 67

A mutagenic effect of allylisothiocyanate (AITC) onE. coli WP 67 (trp uvrA polA) was only found in a test with metabolic activationin vitro. Mixed function oxidases containing cytochrome P-450 are not responsible for the conversion of AITC to mutagenic products. Liver microsomal fractions of rat, mouse, chinese hamster, goat and monkey were used forin vitro metabolic activation. The mutagenic effect of AITC differed in individual animal species according to the protein content in the microsomel fraction. Allylamine (formed by hydrolysis of AITC) did not exhibit any mutagenic effect.     

Metabolic activation of dimethylnitrosamine and diethyl-nitrosamine to mutagens

Dimethylnitrosamine (DMN) and diethylnitrosamine (DEN) are not mutagenic by themselves, but they can be converted by mammalian enzymes to highly mutagenic products. As indicators for mutagenic activity, Neurospora crassa and Salmonella typhimurium were used. The ad-3 forward-mutation system was used to detect specific locus mutations; mutants in this system can range from multi-locus deletions to leaky mutations. The induction of mutations in S. typhimurium is detected as induction of histidine revertants of the histidine-requiring strain G₄6. The activation of DMN is microsomal, inhibited by SKF 525-A, and requires co-factors. The activating enzyme is induced in mice by pretreatment with phenobarbital, 3-methylcholanthrene and butylated hydroxytoluene. The mutagenic activity of the reaction products is directly correlated with the metabolic formation of formaldehyde with and without induction by 3-methylcholanthrene and across strains of mine. Formaldehyde does not contribute to the mutagenic activity of the reaction products. It is clear from the data that the reversion sites in G₄6 are more sensitive than the ad-3 loci of Neurospora crassa to the mutagenic action of DMN metabolites formed by mammalian liver. The microsomal assay is a few orders of magnitude more sensitive than the intraperitoneal host-mediated assay, and the intrahepatic host-mediated assay is a few orders of magnitude more sensitive than the in vitro microsomal system.     

Evaluation of the mutagenic effect of the new fungicide trimorphamide

The new Czechoslovak fungicide trimorphamide was tested for its mutagenic activity. To evaluate the potential mutagenic effects on Drosophila, trimorphamide at 0.5, 1.0, 5.0, 10.0% was administered into the cultivation medium, and the sex-linked recessive lethal mutation detection test and the chromosome nondisjunction test were used. After administration of trimorphamide to mice at 60, 150 and 300 mg . kg-1 b.w. perorally, and 30, 70 and 150 mg . kg-1 b.w. intraperitoneally in single and repeated (5X) doses, a cytogenetic analysis of chromosomal aberrations in bone-marrow cells was performed. The cytogenetic analysis of human peripheral lymphocytes for chromosomal aberrations in vitro was performed 24 h after trimorphamide had been applied into the culture in concentrations 19.1 X 10(-3), 19.1 X 10(-4) and 19.1 X 10(-5) M. Under our testing conditions the trimorphamide concentrations used did not show any mutagenic effect upon Drosophila, compared with the controls. Also, under the conditions of the cytogenetic analysis, no significant increase in the frequency of chromosomal abnormalities in mouse bone marrow or in human peripheral lymphocyte was observed compared with the group of controls.     

Mutagenicity of organophosphorus compounds in bacteria and drosophila

140 Organophosphorus compounds (OP's) have been tested for mutagenic activity in bacteria, principally by using two specially constructed sets of tester strains of the bacteria Salmonella typhimurium and Escherichia coli. It was found that 20% gave positive mutagenic responses and that this group of chemicals produce base substitutions rather than frame-shift mutations. In most cases the DNA repair genes exrA⁺ and recA⁺ were for mutagenic activity.Seven compounds were further tested in Drosophila melanogaster for the ability to induce recessive lethal mutations. In some of these cases the doses administered to the flies had to be very low due to the highly toxic nature of the compounds. To overcome this problem, the accumulation of recessive lethal mutations was measured in populations which were continually exposed to the compounds over a period of some 18 months. During this time the populations developed increased resistance to the compound and so the dose administered could gradually be increased. Six of the compounds were mutagenic.Of the compounds tested in both systems, those showing mutagenic activity in bacteria were also mutaganic in Drosophila, those mutagenic in bacteria were not mutagenic in Drosophila.     

Integrated control of fruit rot and powdery mildew of chilli using the biocontrol agent Pseudomonas fluorescens and a chemical fungicide

A combined strategy of chilli fruit rot and powdery mildew control consisting of reduced fungicide dose and biological control with antagonistic Pseudomonas fluorescens (Pf1) was evaluated. The sensitivity of P. fluorescens to fungicide azoxystrobin at different concentrations (100, 150, 200, 250 and 300 ppm) was tested by turbidometric method. The grown bacterium (Pf1) was tolerant to all concentrations of azoxystrobin. In two field trials, Pf1 tested in combination with reduced concentration of azoxystrobin was highly efficient in management of both diseases of chilli. Biological control of Colletotrichum capsici and Leveillula taurica with P. fluorescens (Pf1) was effective but less so than fungicide alone at the standard dose. However, combination of the biological control agent with a 50% reduction of fungicide dose was as effective as the standard fungicide alone. Application of P. fluorescens along with azoxystrobin significantly increased the survival of Pf1 in the phylloplane of chilli. Further, there were ‘twofold’ increases in activities of peroxidase, polyphenol oxidase, phenylalanine ammonia-lyase, β-1,3-glucanase, chitinase and phenolics in plants treated with Pf1 plus azoxystrobin.     

Evaluation of mutagenic effect of the fungicide fenaminosulf in Drosophila melanogaster

Fenaminosulf (p-dimethylaminobenzenediazo sodium sulfonate, CAS registry No. 140-56-7) which is an active ingredient in several commercial fungicides was reported to be mutagenic in Salmonella typhimurium (McCann et al., 1975), Bacillus subtilis (Kada et al., 1974) and shown to cause chromosome aberrations in plants (Zutshi and Kaul, 1975). Since fenaminosulf has structural similarity to the potent carcinogen, butter yellow (p-dimethylaminoazobenzene, CAS registry No. 60-11-7), the present studies were undertaken to evaluate the mutagenic potential of this fungicide in Drosophila melanogaster. Fenaminosulf administered at 10 mg/100 ml food medium failed to induce sex-linked recessive mutations in Drosophila. Since Drosophila has drug-metabolizing enzymes similar to those of mammals (Vogel, 1975), it is suggested that the lack of mutagenic activity of fenaminosulf could be due to the conversion of fenaminosulf to non-mutagenic derivatives in Drosophila.     

5-Methyltryptophan resistance mutations in Escherichia coli K-12. Mutagenic activity of monofunctional alkylating agents including organophosphorus insecticides

The induction of 5-methyltryptophan (5-MT) resistance mutations was assayed as a test system for mutagenic chemicals in Escherichia coli. It is assumed that different premutational alterations in several genes of the Escherichia coli chromosome will lead to 5-MT-resistant mutants. The chemicals used were three monofunctional alkylating agents as reference compounds, namely β-propiolactone (β-PL), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), and methyl methanesulfonate (MMS), which are all mutagenic in the 5-MT system; of the eight organophosphorus insecticides tested, four have definite mutagenic activity (Dichlorvos, Oxydemetonmethyl, Dimethoate, and Bidrin), one is probably mutagenic (Methylparathion) and the remaining three (Parathion, Malathion and Diazinon) do not induce 5-MT resistance mutations in the conditions used here (< 30% survival). The relative mutagenic activity after a treatment time of 60 min is (in decreasing order) MNNG > MMS > Dichlorvos > Oxydemetonmethyl, Dimethoate and Bidrin. The concentration-dependent mutagenic activity of all mutagenic compounds is nearly linear when plotted on a log-log scale (with slopes varying from 1.0 to 1.5) and could be taken as an indication that one premutational reaction will be sufficient for the induction of one 5-MT-resistant mutant.     

Novel benzofuran constituent from the husk of Carya cathayensis Sarg.

In an attempt to find the effective botanical fungicide, investigations were conducted on the husk of Carya cathayensis Sarg. (CCS). The results suggested that the crude extracts from the husk of CCS have obvious germicidal effects on the pathogenic fungi, while screening for new antifungal agents from the husk of C. cathayensis Sarg., a novel benzofuran-type compound, cathayenone A, was obtained. Its structure was elucidated by 1-D and 2-D NMR and MS data analyses. The inhibition rates of 0.1 mg/mL of cathayenone A against the spore germination of Exserohilum turcicum and Pyricularia oryzae were 95.8% and 86.7%, respectively, which showed better antifungal activity than tebuconazole fungicide. Therefore, cathayenone A has the potential to be a fungicide.     

At Least Two Origins of Fungicide Resistance in Grapevine Downy Mildew Populations

Quinone outside inhibiting (QoI) fungicides represent one of the most widely used groups of fungicides used to control agriculturally important fungal pathogens. They inhibit the cytochrome bc₁ complex of mitochondrial respiration. Soon after their introduction onto the market in 1996, QoI fungicide-resistant isolates were detected in field plant pathogen populations of a large range of species. However, there is still little understanding of the processes driving the development of QoI fungicide resistance in plant pathogens. In particular, it is unknown whether fungicide resistance occurs independently in isolated populations or if it appears once and then spreads globally by migration. Here, we provide the first case study of the evolutionary processes that lead to the emergence of QoI fungicide resistance in the plant pathogen Plasmopara viticola. Sequence analysis of the complete cytochrome b gene showed that all resistant isolates carried a mutation resulting in the replacement of glycine by alanine at codon 143 (G143A). Phylogenetic analysis of a large mitochondrial DNA fragment including the cytochrome b gene (2,281 bp) across a wide range of European P. viticola isolates allowed the detection of four major haplotypes belonging to two distinct clades, each of which contains a different QoI fungicide resistance allele. This is the first demonstration that a selected substitution conferring resistance to a fungicide has occurred several times in a plant-pathogen system. Finally, a high population structure was found when the frequency of QoI fungicide resistance haplotypes was assessed in 17 French vineyards, indicating that pathogen populations might be under strong directional selection for local adaptation to fungicide pressure.     

Investigation of styrene in the liver perfusion/cell culture system. No indication of styrene-7,8-oxide as the principal mutagenic metabolite produced by the intact rat liver

Mutagenic effect of styrene and styrene-7,8-oxide was studied with the isolated perfused rat liver as metabolizing system and Chinese hamster V79 cells as genetic target cells. Styrene-7,8-oxide which is mutagenic per se was rapidly metabolized by the perfused rat liver. Thus no mutagenic effect was detected neither in the perfusion medium nor in the bile. However when styrene was added to the perfusion system, an increase in V79 mutants was observed regardless of where in the circulating perfusion medium the V79 cells were placed: the same effect was obtained with V79 cells close to the liver as well as at a distance from the liver. No mutagenic effect was observed in the bile. Simultaneous analysis of the styrene-7,8-oxide concentration in the perfusion medium, suggest that this metabolite is not the cause of the mutagenic effect observed during perfusion with styrene.The effect of the two test compounds on some liver functions was also studied. Both styrene and styrene-7,8-oxide changed the bile flow without affecting bile acid secretion: styrene caused a reduction in bile flow as compared to control perfusions and styrene-7,8-oxide increased the bile flow. Styrene, but not styrene-7,8-oxide, reduced gluconeogenesis from lactate. Styrene had no effect on the liver's capacity to incorporate amino acids into plasma proteins, whereas styrene-7,8-oxide reduced the amino acid incorporation. The microsomal cytochrome P-450 content was not affected by the two test compounds. No alteration in microsomal N- and C-oxygenation of N, N-dimethylaniline (DMA) was observed with styrene-7,8-oxide or the lower styrene dose used (240 μmol), whereas the higher styrene concentration (480 μmol) reduced N-oxygenation and thus also the total DMA metabolism.It is suggested that the results on styrene and styrene-7,8-oxide found here using the liver perfusion/cell culture system mimic the metabolism expected to be found in the intact animal, thus indicating that styrene-7,8-oxide is not the principal mutagenic metabolite of styrene in vivo.     

Ribozyme-mediated REV1 inhibition reduces the frequency of UV-induced mutations in the human HPRT gene

In yeast, mutations induced by UV radiation are dependent on the function of the Rev1 gene product, a Y-family DNA polymerase that assists in translesion replication with potentially mutagenic consequences. Human REV1 has been cloned, but its role in mutagenesis and carcinogenesis remains obscure. To examine the role of REV1 in UV mutagenesis in human cells and to evaluate its potential as a therapeutic target to prevent such mutations, we developed a ribozyme that cleaves human REV1 mRNA in vitro. Stable expression of the ribozyme in human cells reduced the target REV1 mRNA up to 90%. We examined the cytotoxic and mutagenic response to UV of seven independent clones that had reduced levels of endogenous REV1 mRNA. In each case, the clonogenic survival after UV was not different from that of the parental cell strains. In contrast, the UV-induced mutant frequencies at the endogenous HPRT locus were reduced up to 75% in cells with reduced levels of REV1 mRNA. The data support the idea that targeting the mutagenic translesion DNA replication pathway can greatly reduce the frequency of induced mutations.     

Involvement of the L-cysteine biosynthetic pathway in azide-induced mutagenesis in Salmonella typhimurium

L-Cystine and L-cysteine specifically reverse the mutagenic action of azide in Salmonella typhimurium and Escherichia coli. To establish whether the L-cysteine biosynthetic pathway is involved in azide-induced mutagenesis, several derivatives of a mutagen tester-strain of S. typhimurium bearing mutations in different cys genes were isolated. No mutagenic effect of azide was observed in a strain carrying mutation in the cysE gene, unless the incubation medium was supplemented with exogenous O-acetylserine. Out of 16 cysK mutants 14 were mutagenized by azide very poorly or not at all. These results indicate that the activity of O-acetylserine sulfhydrylase A, and the availability of O-acetylserine, one of the two co-substrates of the enzyme, are essential for the mutagenic action of azide in S. typhimurium     

Mutagenic effect of nalidixic acid on bacteriophage T4 (author's transl)

Nalidixic acid can efficiently induce the reversion of some T4 rII mutations. The great majority of the strains whose reversion can be induced by this antibiotic are also sensitive to the mutagenic action of proflavin, indicating that mutagenicity of nalidixic acid results in base pair addition or deletion. With bacterial host strains resistant to nalidixic acid, the mutagenic effect is greatly reduced but not the effects on phage multiplication. This fact shows that the mutagenic agent is not the nalidixic acid itself but a derivative synthesized in sensitive bacterial strains.     

Mutagenic activity of the methyl and phenyl derivatives of the food mutagen 2-amino-3-methylimidazo[4,5-f]quinoxaline (IQx) in the Ames test

The mutagenic activity of 15 different mono-, di-, tri-, and tetramethyl derivatives of the food mutagen IQx (2-amino-3-methylimidazo[4,5-f]quinoxaline), one diphenyl derivative of IQx and two phenyl derivatives of 5-MeIQx (2-amino-3,5-dimethylimidazo[4,5-f]quinoxaline) were studied in the Ames test with Salmonella typhimurium TA98 and enzymatic activation (S9). The number and positioning of the methyl groups strongly affected the mutagenic activity. The phenylated compounds showed weak mutagenic potency. It seems that both resonance stabilization of the nitrenium ion and steric effects are important in determining mutagenic potency.     

The influence of the fungicide folcidin on the distribution and metabolism of32P in gherkin plants

High accumulation of³²P was observed in the leaves of intact gherkin plants 9 days after their roots had been treated with 0.005% suspension of the systemic fungicide Folcidin 50WP (cypendazole),i.e. 8 days after the roots had been exposed to labelled phosphate. Folcidin also influenced phosphorus metabolism in the plants. High biologic cytokinin-like activity of the fungicide was established when using a callus cytokinin bioassay.     

Effect of benomyl on spores of Fusarium oxysporum

Benomyl, methyl-1-(butylcarbamoyl)-2-benzimidazole carbamate, inhibited germination of Fusarium oxysporum. The fungicide was found to be very rapidly absorbed by the conidia. Benomyl decreased the RNA content and at high concentrations the respiration rate was inhibited. Among various respiratory enzymes that have been tested, l-malate:NAD oxidoreductase was the most sensitive to the fungicide. Protein content and amino acid uptake did not appear to be significantly altered by treatment with benomyl.     

Differential responses of N-nitrosoamines and aromatic amines in the plant cell/microbe coincubation assay

The plant cell/microbe coincubation assay is based on employing living tobacco cells in suspension culture as the activating system for promutagens and the Ames/Salmonella cells as the genetic indicator system. In contrast to aromatic amines(e.g. 2-aminofluorene andm-phenylenediamine) that were previously reported to be activated to products mutagenic in theS. typhimurium strains TA98 or YG1024 by tobacco cells, promutagenic N-nitrosoamines (N-nitrosodimethylamine, N-nitroso-morpholine, N-nitrosopiperidine, N-nitrosomethyl-2-hydroxypropylamine) were not activated to product(s) mutagenic inS. typhimurium TA 100.